We study regulation of amino acid biosynthetic genes in budding yeast as means of dissecting molecular mechanisms of gene regulation at the translational and transcriptional levels. Transcription of these unlinked genes is coordinately induced by the transcriptional activator GCN4 in response to starvation for any amino acid. Expression of GCN4 is coupled to amino acid levels by a translational control mechanism involving four short upstream open reading frames (uORFs) in GCN4 mRNA. Ribosomes translate the 5-most uORF (uORF1), resume scanning downstream, and under nonstarvation conditions, reinitiate translation at uORFs 2, 3, or 4 and then dissociate from the mRNA, keeping GCN4 translation repressed. Under starvation conditions, the reinitiating ribosomes bypass uORFs 2-4 and reinitiate at the GCN4 start codon instead. This bypass is triggered by decreased formation of the ternary complex (TC), eIF2-GTP-Met-tRNAiMet, which delivers Met-tRNAiMet to the 40S ribosome in assembling the 43S preinitiation complex (PIC). TC abundance is reduced in starved cells by phosphorylation of the alpha subunit of eIF2 (eIF2a) by the protein kinase GCN2, conserved in all eukaryotes, converting eIF2 from substrate to inhibitor of guanine nucleotide exchange factor (GEF), eIF2B. Hence, GCN4 translation is a sensitive in vivo indicator of impaired TC loading on 40S subunits. We previously exploited this fact to isolate mutations in subunits of eIF2B that constitutively derepress GCN4 (Gcd- phenotype) by lowering TC assembly in the absence of eIF2 phosphorylation by GCN2. We recently applied the Gcd- selection to obtain the first functional evidence implicating 18S rRNA residues of the 40S subunit in TC loading and AUG selection. Mutations that perturb the location or identity of the bulge G residue in helix 28 impair TC loading during reinitiation on GCN4 mRNA and, uniquely, increase the bypass of AUG start codons during primary initiation (leaky scanning). At least one such mutation impairs the rate and stability of TC loading on mutant 40S subunits in vitro. Site directed mutations in other residues predicted to contact the AUG codon or anticodon loop of tRNAiMet confer dominant Gcd- and recessive-lethal phenotypes, implying that bacterial elongation complexes are relevant to the structure of eukaryotic PICs. We showed previously that inhibition of eIF2B by phosphorylated eIF2 requires interaction of the S1 domain of eIF2a with a subcomplex of eIF2B formed by the three regulatory subunits, a/GCN3, b/GCD7, and d/GCD2, which blocks the GEF function of the catalytic (e) subunit of eIF2B. The eIF2B regulatory subunits are related in sequence to one another and numerous archaeal proteins, some of which function in methionine salvage and CO2 fixation. Our sequence analyses predicted that members of one phylogenetically distinct group of these archaeal proteins (designated aIF2Bs) are functional homologues of the three regulatory subunits of eIF2B. Supporting this, we have shown that three such aIF2B proteins bind to the a subunit of aIF2 from the cognate Archaeon, and one such aIF2B also binds the S1 domain of yeast eIF2a and interacts with eIF2Ba/GCN3 in yeast cells. Mass spectrometry identified several proteins that copurify with aIF2B from the Archaeon Thermococcus kodakaraensis, including aIF2a, a sugar-phosphate nucleotidyltransferase with sequence similarity to the catalytic subunit eIF2Be, and several large subunit (50S) ribosomal proteins. Based on our evidence that aIF2B has functions in common with eIF2B regulatory subunits, the known crystal structure of aIF2B was used to construct a model of the eIF2B regulatory subcomplex, in which evolutionarily conserved regions and sites of regulatory mutations in eIF2B subunits are juxtaposed in one continuous binding surface for phosphorylated eIF2a. Our results provide important structural insights into the inhibition of eIF2B by phosphorylated eIF2a, the principal means of down-regulating translation during starvation or stress in eukaryotes. The GCN2 kinase domain (KD) is inert and must be activated by binding of uncharged tRNA, which accumulates in starved cells, to the adjacent regulatory domain. We showed previously that Gcn2 latency results from inflexibility of the hinge connecting N- and C-lobes and a partially obstructed ATP-binding site in the KD. We have now demonstrated that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining rotation of helix alphaC (aC), in a manner relieved by tRNA binding and autophosphorylation of Thr-882 in the KD activation loop. Mutationally disrupting the hydrophobic network constitutively activates eIF2a phosphorylation and bypasses the requirement for tRNA binding and Thr-882 autophosphorylation. In particular, replacing Leu-856 with any non-hydrophobic residue activates Gcn2, while hydrophobic amino acid substitutions maintain latency. We further show that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856-hydrophobic network or enhance hinge flexibility eliminate the need for the parallel dimer interface, implying that KD dimerization facilitates reorientation of aC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of aC are mutually reinforcing conformational transitions stimulated by tRNA binding and locked in by autophosphorylation of T882. Our results have implications for the latency mechanisms of other important protein kinases, including PKR, the EFG receptor and CDK2. In the arena of transcriptional control, we showed previously that activation by GCN4 depends on its recruitment of the coactivators Mediator, SAGA, SWI/SNF, and RSC, which mediate nucleosome remodeling and recruitment of general transcription factors and RNA Polymerase II (Pol II) to promoters. More recently, we discovered that SAGA is recruited co-transcriptionally to coding sequences by association with the Ser5-phosphorylated CTD of Pol II, and that the histone acetyltransferase subunit of SAGA (GCN5) promotes histone eviction, Pol II processivity, and efficient histone H3-Lys4 methylation in coding sequences. In collaboration with the Rodriguez-Navarro group in Valencia, we further showed that SUS1, a subunit of both SAGA and mRNA export complex TREX2, is associated with elongating Pol II and essential mRNA export factors, and that SUS1 promotes transcription elongation, demonstrating that SUS1 acts at the interface between SAGA and TREX2 to coordinate transcription elongation with mRNA export. The cyclin-dependent kinase BUR1/BUR2 appears to be the yeast ortholog of mammalian P-TEFb, which phosphorylates Ser2 of the Pol II CTD, but the importance of BUR1/BUR2 in CTD phosphorylation was unclear. We have now shown that BUR1/BUR2 is co-transcriptionally recruited to the 5 end of the Gcn4 target gene ARG1 in a manner stimulated by interaction of the BUR1 C-terminus with CTD repeats Ser5-phosphorylated by the KIN28 subunit of TFIIH. Remarkably, impairing BUR1/BUR2 function, or removing the CTD-interaction domain in BUR1, reduces Ser2 phosphorylation in bulk Pol II and eliminates the residual Ser2P in cells lacking the major Ser2 CTD kinase, CTK1. Impairing BUR1/BUR2 or CTK1 evokes a similar reduction of Ser2P in total elongating Pol II molecules phosphorylated on Ser5, and in elongating Pol II near the ARG1 promoter. By contrast, CTK1 is responsible for the bulk of Ser2P in total Pol II and at promoter-distal sites. Our findings indicate that BUR1/BUR2 is recruited to promoters by Pol II phosphorylated on Ser5 by TFIIH, where it then directly phosphorylates the CTD on Ser2 and also stimulates Ser2 phosphorylation by CTK1.